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Phytochemical screening and antimicrobial activity testing of crude

hydroalcoholic extract from leaves of Sphagneticola trilobata (Asteraceae)

Article in Ciência Rural · April 2019

DOI: 10.1590/0103-8478cr20180639

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Ciência Rural,
Phytochemical Santaand
screening Maria, v.49:04,
antimicrobial e20180639,
activity 2019hydroalcoholic extract fromhttp://dx.doi.org/10.1590/0103-8478cr20180639
testing of crude leaves of Sphagneticola trilobata (Asteraceae).
ISSNe 1678-4596
MICROBIOLOGY

Phytochemical screening and antimicrobial activity testing of crude hydroalcoholic

extract from leaves of Sphagneticola trilobata (Asteraceae)

Ana Greice Borba Leite1 Eulina Tereza Nery Farias1 Ana Paula de Oliveira2
Ruan Emmanuell Franco de Abreu3 Mateus Matiuzzi da Costa3
Jackson Roberto Guedes da Silva Almeida2 Lígia Reis de Moura Estevão1
Joaquim Evêncio-Neto1*
1
Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco (UFRPE), 52171-900, Recife, PE, Brasil.
E-mail: joaquim.evenciont@ufrpe.br. *Corresponding author.
2
Centro de Estudos e Pesquisas em Plantas Medicinais, Central de Análises de Fármacos, Medicamentos e Alimentos, Universidade Federal do
Vale do Rio São Francisco (UNIVASF), Petrolina, PE, Brasil.
3
Universidade Federal do Vale do Rio São Francisco (UNIVASF), Petrolina, PE, Brasil.

ABSTRACT: This study aimed to perform phytochemical analysis and to test the antimicrobial activity of the crude hydroalcoholic extract
obtained from the leaves of Sphagneticola trilobata. Classes of secondary metabolites present in the extract were identified through phytochemical
screening using analytical thin-layer chromatography. Antimicrobial activity was evaluated by testing cultures of Staphylococcus aureus,
S. epidermidis, Staphylococcus spp., Escherichia coli, Serratia marcescens, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella
Typhimurium, and Klebsiella pneumoniae isolated from human skin and those of Staphylococcus spp. isolated from dog skin using the broth
microdilution method. In the phytochemical screening, classes of anthracenic derivatives and mono-, sesqui-, and diterpenes were identified.
Colorimetric analysis showed total phenol and total flavonoid contents of 21.7 ± 0.009 mg of gallic acid equivalents per gram of sample and
0.23 ± 0.005 mg of catechin equivalents per gram of sample, respectively. Microbiological analysis revealed that the hydroalcoholic extract of
S. trilobata exhibited antimicrobial activity against cultures of Staphylococcus spp., E. coli, S. marcescens, and E. faecalis isolated from human
skin and those of Staphylococcus spp. isolated from dog skin. Thus, crude hydroalcoholic extract of leaves of S. trilobata contained flavonoids
and terpenoids as secondary metabolites, which contributed to its antimicrobial activity against skin bacteria isolated from different sources.
Key words: Antimicrobial, Phytochemical Screening, Phenolic compounds.

Triagem fitoquímica e atividade antimicrobiana do extrato hidroalcoólico bruto

das folhas de Sphagneticola trilobata (Asteraceae)

RESUMO: Este estudo teve como objetivo realizar a triagem fitoquímica preliminar e testar a atividade antimicrobiana do extrato
hidroalcoólico bruto das folhas de Sphagneticola trilobata. A identificação das classes de metabólitos secundários presentes no extrato foi
realizada através da cromatografia em camada delgada analítica (CCDA). Para determinar a quantidade de fenóis e flavonoides totais
foram utilizados os métodos espectrofotométricos de Folin-Ciocalteu e complexação com AlCl3, respectivamente. Para avaliar a atividade
antimicrobiana foram testadas culturas de Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus spp., Escherichia coli, Serratia
marcescens, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella Typhimurium, Klebsiella pneumoniae isoladas de pele humana e
culturas de Staphylococcus spp. isoladas de pele de cães pelo método de microdiluição em caldo. Na triagem fitoquímica foi verificada
reação positiva para a presença de derivados antracênicos, mono, sesqui e diterpenos. As análises colorimétricas mostraram conteúdos de
fenóis totais e flavonoides totais de 21,7 ± 0,009 miligramas de equivalentes de ácido gálico por grama de amostra e 0,23 ± 0,005 miligramas
de equivalentes de catequina por grama de amostra, respectivamente. Na análise microbiológica, o extrato hidroalcoólico das folhas de
Sphagneticola trilobata apresentou atividade antimicrobiana frente às culturas de Staphylococcus spp., Escherichia coli, Serratia marcescens
e Enterococcus faecalis. Todas as culturas de Staphylococcus spp. isoladas de pele de cães foram sensíveis ao extrato. Conclui-se que o
extrato hidroalcoólico bruto das folhas de Sphagneticola trilobata possui entre seus metabólitos secundários os flavonoides e terpenoides que
contribuíram com a atividade antimicrobiana frente às bactérias isoladas de pele de diferentes origens.
Palavras-chaves: antibacteriano, estudo fitoquímico, compostos fenólicos.

INTRODUCTION and pharmacological studies to determine the efficacy

Two decades ago, CECHINEL-FILHO &
YUNES (1998) reported advances in phytotherapy
and growth in the market for phytotherapeutic agents,
highlighting the importance of conducting chemical
Received 08.08.18
of medicinal plants. Several medicinal plants are used
without particularly confirming their pharmacological
properties, which raises the possibility of these plants
being harmful to health due to their adverse effects
and toxicity (VEIGA JÚNIOR et al., 2005).
Approved 02.22.19 Returned by the author 03.01.19
CR-2018-0639.R2 Ciência Rural, v.49, n.4, 2019.
2 Leite et al.
Sphagneticola trilobata is a plant that
has been very commonly used in folk medicine
in Brazil; however, few studies have evaluated its
activity. It is a herbaceous plant from the Asteraceae
family and is popularly known as malmequer,
vedélia, and picão-da-praia in Brazil. It is native to
Brazil, easily grows, and quickly spreads on several
different types of soil. It grows well both under the
sun and in the shade and can easily be found in dark
places, on seashores, and in vacant lots, forming a
carpet of foliage (CORREIA, 1984).
Although, phytochemical analysis studies
of S. trilobata have been conducted (CECHINEL
FILHO, 2000; CARVALHO et al., 2001; FIDELIS
et al., 2005; SILVA et al., 2012; SHANKAR &
TOMAS, 2014), it is important to identify the
chemical composition of extracts from this plant
collected from different regions of Brazil because
the composition may vary with changes in soil
and climate. Thus, the antimicrobial activity of the
extract from this plant against bacteria isolated from
human and animal skin is directly related to the
variation in its chemical components under different
environmental conditions.
Therefore, this study aimed to perform a
preliminary phytochemical screening and to evaluate
the antimicrobial activity of hydroalcoholic extract
from the leaves of S. trilobata against skin isolates
of Gram-positive and Gram-negative bacteria from
different sources.
MATERIALS AND METHODS
Seven samples of the plant species were
collected at blooming from the campus of the Federal
Rural University of Pernambuco (UFRPE) located at
geographic coordinates 8° 04’ 03” S and 34° 55’ 00” W,
in the morning in May 2015. The material was sent to
the Geraldo Mariz Herbarium of the Federal University
of Pernambuco, where the botanical dehydrated
material was deposited, identified, and catalogued
under the registration number 78.782.
To produce the hydroalcoholic extract, the
plant’s leaves were dehydrated in an oven at 40°C and
then ground and macerated in 70% ethanol for 72 h. The
extract/plant proportion was 2 mL of the solvent—70%
ethanol (Merck ethanol PA)—for 1 g of plant material,
according to the method described by MATOS (1997).
The extracting solution was filtered and then concentrated
in a rotary evaporator under reduced pressure at 45°C,
which produced a crude hydroalcoholic extract with
a concentration of 140 mg/mL. Procedures were
performed at the UFRPE Laboratory of Pharmacology.
Phytochemical assays were performed at the
Center for Studies and Research on Medicinal Plants
of the University of the São Francisco River Valley
(NEPLAME-UNIVASF). Preliminary phytochemical
screening was performed using analytical thin-layer
chromatography. The extract was applied with a glass
capillary tube onto plates with an aluminum support
using silica gel 60 F254 as an adsorbent and then eluted
using different solvent systems, as described by
WAGNER & BLADT (1996).
For determining the total phenol content, an
aliquot (40 µL) of the extract was added to 3.16 mL of
distilled water and 200 µL of Folin– Ciocalteu reagent,
mixed immediately in succession. The mixture was
made to stand for 6 min, and 600 µL of a stock Na2CO3
solution was then added. The final solutions were
made to stand at 20°C for 2 h. At the end of the process,
the absorbance of each solution was determined
using a spectrophotometer at 756 nm against blank
(all components, except the sample being analyzed),
and results were plotted on a graph correlating the
absorbance of the samples with their concentration.
The total phenolic content of extracts was expressed
as milligrams of gallic acid equivalents per gram of
sample (mg EqGA/g) using the gallic acid calibration
curve, which was obtained in concentrations ranging
from 50 to 1,000 mg/L (SLINKARD & SINGLETON,
1977). All assays were performed in triplicate.
Total flavonoid content was determined
using the metallic complexation method previously
described by ZHISHEN et al. (1999). First, 300 µL
of the crude hydroalcoholic extract and the same
volume of (+)-catechin standard solution were added
to 1.5 mL of distilled water, following which 90
µL of NaNO2 solution was added. After letting the
mixture react for 6 min, 180 µL of 10% AlCl3·H2O
solution was added to it. After reacting for 5 min, 600
µL of 1 M NaOH solution was added to the previous
mixture. Finally, the volume was topped with 330
µL of distilled water, and the system was completely
homogenized. Immediately after obtaining the final
mixture, absorbance was measured against the blank
at 510 nm on a spectrophotometer and compared with
standard solutions containing (+)-catechin at known
concentrations. Results were expressed as milligrams
of catechin equivalents per gram of sample (mg
EqC/g) by comparison with the standard catechin
curve, which was obtained in concentrations ranging
from 50 to 1,000 mg/L. All assays were performed in
triplicate (ZHISHEN et al., 1999).
Antimicrobial activity was evaluated using
the broth microdilution method at the Federal University
of the São Francisco River Valley (UNIVASF)
Ciência Rural, v.49, n.4, 2019.
Phytochemical screening and antimicrobial activity testing of crude hydroalcoholic extract from leaves of Sphagneticola trilobata (Asteraceae).

Laboratory of Microbiology and Animal Immunology. change in the color of TTC from colorless to red
First, 0.25 mg of the crude hydroalcoholic extract from indicates bacterial metabolic activity. All assays were
the leaves of S. trilobata was weighed and diluted performed in triplicate (CLSI, 2014).
in alcohol and water (3:7), which resulted in a stock
solution with a concentration of 25,000 μg/mL. Fifteen RESULTS
isolates of Gram-positive and Gram-negative bacteria
from different sources, including isolates from human Phytochemical screening of the crude
skin, such as S. aureus (ATCC 25923), S. epidermidis hydroalcoholic extract from leaves of S. trilobata
(ATCC 12228), Staphylococcus spp. (MRSA 8536 and revealed the presence of phenolic compounds,
MRSA 9606), E. coli (ATCC 35218), S. marcescens anthracene derivatives, and mono-, sesqui-, and
(ATCC 13880), E. faecalis (ATCC 19433), P. diterpenes (Table 1). The total phenol and total
aeruginosa (ATCC 27853), S. Typhimurium flavonoid content was 21.7 ± 0.009 mg EqGA/g and
(ATCC 10708 and ATCC 14023), and K. pneumoniae 0.23 ± 0.005 mg EqC/g, respectively.
(ATCC 13883), and those from dog skin, including The following bacterial cultures isolated
four strains of Staphylococcus spp. (244, 246, 250, from human skin exhibited susceptibility to the crude
and 256) during clinical consultations at the UNIVASF hydroalcoholic extract from the leaves of S. trilobata:
University Veterinary Hospital. Bacterial isolates used Staphylococcus spp. (MRSA 8536 and MRSA 9606),
in this experiment were chosen for their importance E. coli (ATCC 35218), S. marcescens (ATCC 13880),
in several dermatological diseases occurring in both and E. faecalis (ATCC 19433) (Table 2).
human and veterinary clinical practice. The strains Some cultures exhibited resistance to the
were available at the bacteria collection of the extract, including P. aeruginosa (ATCC 27853), S.
UNIVASF Laboratory of Microbiology and Animal Typhimurium (ATCC 10708 and ATCC 14023), and
Immunology. All isolates were reseeded in trypticase K. pneumoniae (ATCC 13883).
soy agar medium for 24 h at 37°C. All bacterial cultures isolated from dog skin
For preparing bacterial inoculum, four obtained from the UNIVASF University Veterinary
colonies were inoculated into tubes containing 5 mL Hospital, i.e., Staphylococcus spp. (244, 246, 250, and
of saline until the McFarland turbidity scale value of 256), were susceptible to the crude hydroalcoholic
0.5 was obtained. Then, 0.1 mL of the suspension was extract from the leaves of S. trilobata (Table 2).
inoculated into tubes containing 9.9 mL of Mueller–
Hinton broth. In a 96-well microplate, 200 μL of DISCUSSION
Mueller–Hinton broth was added to each well, and a
serial dilution was performed in the same wells starting S. trilobata contains several classes of
with 200 μL of the extract followed by a 1:2 dilution, secondary metabolites exhibiting pharmacological
discarding the final 200 μL. The serial dilutions were
tested at the following final concentrations: 12,500,
6,250, 3,125, 1,562.5, 781.3, 390.6, 195.3, and 97.6 μg/
mL. Wells were then inoculated with 20 μL of the Table 1 - Determination of compounds present in the crude
Mueller–Hinton broth containing the tested bacterium, hydroalcoholic extract from the leaves of
Sphagneticola trilobata by phytochemical
separately in each well. The last wells received the screening.
diluent control (the diluent plus Mueller–Hinton
broth and the tested bacterium), positive control (the Class Result
Mueller–Hinton broth and the tested bacterium), and
Alkaloids –
negative control (the Mueller–Hinton broth alone). Anthocyanins –
Plates were incubated in a bacteriological oven at Anthraquinones –
37°C, and the growth conditions of microorganisms Flavonoids ++
Coumarins –
were checked after 24 h (CLSI, 2014).
Anthracene derivatives +
The antimicrobial activity was evaluated Lignans –
considering the most dilute concentration of the crude Saponins –
hydroalcoholic extract that inhibited bacterial growth Condensed tannins –
Hydrolyzable tannins –
in the tested tube (minimum inhibitory concentration, Triterpenes and steroids –
MIC), assessed by a negative result in the colorimetric Mono-, sesqui-, and diterpenes +
reaction produced by adding 10 μL of 2,3,5-triphenyl
tetrazolium chloride (TTC) at 2% to each well. A (–) negative; (+) weakly positive; (++) moderately positive.

Ciência Rural, v.49, n.4, 2019.

4 Leite et al.
properties, including tannins, saponins, flavonoids,
phenolic compounds, and terpenoids (BALEKAR et al.,
2014). SOUZA-MOREIRA et al. (2010) have highlighted
the importance of determining the chemical constituents
of a plant and identifying the active compounds among
them and their possible adverse effects.
BALEKAR et al. (2012) analyzed the
ethanolic extract from the leaves of S. trilobata
collected from the campus of the Prince of Songkla
University in Thailand in February and reported
that the total phenol content was 74.38 ± 1.03 mg/g,
which was measured using the Folin–Ciocalteu
method, and the total flavonoid content was
16.67 ± 0.74 mg/g in quercetin equivalents. Higher
contents obtained in their study than those obtained
in the present study are attributable to the solvent
used in extraction and differences in climate and
soil because the extracts were produced from plants
collected from different locations.
Other studies have analyzed the chemical
composition of the aerial parts of S. trilobata and
identified the main chemical constituents of this
species, such as kaurenoic acid, luteolin, flavonoids,
tannins, and essential oils (CECHINEL FILHO,
2000; FIDELIS et al., 2005; SILVA et al., 2012).
In addition to these, other compounds have been
isolated from the flowers, such as stigmasterol and
its glucosides, β-sitosterol, oleanolic acid ester
derivatives (CARVALHO et al., 2001), phenolic
compounds, flavonoids, and terpenoids (SHANKAR
& TOMAS, 2014).
The presence of these compounds in
the chemical composition of S. trilobata makes it a
widely used species in folk medicine. Phenols and
flavonoids are potent antioxidants (PRIOR & CAO,
Table 2 - Minimum inhibitory concentration of crude hydroalcoholic extract from the leaves of Sphagneticola trilobata against bacterial
cultures isolated from human and dog skin.
Bacterial cultures
Human skin
Staphylococcus aureus (ATCC 25923)
Staphylococcus epidermidis (ATCC 12228)
Staphylococcus spp. (MRSA 8536)
Staphylococcus spp. (MRSA 9606)
Escherichia coli (ATCC 35218)
Serratia marcescens (ATCC 13880)
Enterococcus faecalis (ATCC 19433)
-----------------------------------------------------------------------Dog skin-----------------------------------------------------------------------------------
Staphylococcus spp. (244)
Staphylococcus spp. (246)
Staphylococcus spp. (250)
Staphylococcus spp. (256)
2000; VIEIRA et al., 2015), and flavonoids also
exhibit antimicrobial activity (RAJARATHINAM
& DRONAMRAJU, 2018). Terpenes exhibit anti-
nociceptive, anti-inflammatory, and antimicrobial
properties (SARTORI, 2005), and anthracene
derivatives, the presence of which has not been
reported in this species to date, exhibit astringent
activity (SOUSA et al., 2003).
In addition to phytochemical studies,
advances have been made in research on
pharmacological actions of plants exhibiting
antimicrobial activity with an aim of obtaining
novel compounds with biological activity. Although,
the bactericidal properties of several species have
been empirically acknowledged for centuries, these
properties have started to be scientifically confirmed
only in the last few decades (HAIDA et al., 2007).
One of the bacterial genera tested in the
present study was Staphylococcus spp. The crude
hydroalcoholic extract from the leaves of S. trilobata
inhibited the growth of cultures isolated from human
and dog skin. Similar results were observed in a study
by SARTORI (2005) in which fractions of extracts
from flowers of the same species inhibited the growth
of S. aureus; that study evidenced that the hexane,
dichloromethane, and butanol fractions of the extract
exhibited antimicrobial activity with an MIC between
250 and 1,000 μg/mL. BALEKAR et al. (2012)
observed that the ethyl acetate fraction of the ethanolic
extract from leaves of S. trilobata was active against
S. aureus and S. epidermidis, with an MIC of 62.5 and
31.25 μg/mL, respectively. SHANKAR & TOMAS
(2014) tested the antimicrobial activity of various
extracts from the flowers of S. trilobata against other
bacterial species, such as Vibrio parahaemolyticus,
Minimum inhibitory concentration
3,125 µg/mL
3,125 µg/mL
6,250 µg/mL
6,250 µg/mL
12,500 µg/mL
12,500 µg/mL
6,250 µg/mL
3,125 µg/mL
1,562.5 µg/mL
3,125 µg/mL
6,250 µg/mL
Ciência Rural, v.49, n.4, 2019.
Phytochemical screening and antimicrobial activity testing of crude hydroalcoholic extract from leaves of Sphagneticola trilobata (Asteraceae).
Streptococcus haemolyticus, Proteus rettgeri, and
P. vulgaris, and concluded that the ethanolic extract
from the flowers exhibited antimicrobial activity.
Other studies that reported the bactericidal
activity of medicinal plants and importance of natural
products can easily be reported in the literature, such
as the study conducted by DUARTE (2006) who
observed that extracts from Mikania glomerata and
M. laevigata inhibited S. aureus and S. faecium, with
MIC values between 0.04 and 0.1 mg/mL, which is
similar to the MIC of chloramphenicol (0.12 mg/mL).
PARENTE et al. (2009) observed that the ethanolic
extract from the flowers of Calendula officinalis
L. exhibits antimicrobial activity against S. aureus.
Likewise, HAIDA et al. (2007) observed that the crude
extract from the aerial parts of Rosmarinus officinalis
exhibited inhibitory activity against S. aureus.
E. coli is an important bacterium that is
commonly used to study the antimicrobial activity
of phytotherapeutic products and was found to be
inhibited by the crude hydroalcoholic extract from the
leaves of S. trilobata. Other plants have been studied
and found to exhibit this activity, such as various
extracts from the aerial parts of Bidens pilosa and
Origanum majorana (HAIDA et al., 2007). However,
SILVA & ALMEIDA (2014) did not find the same
result when they tested the crude ethanolic extract
from the bark of Carapa guianensis Aubl.
E. faecalis was another bacterium tested in
the present study with the crude hydroalcoholic extract
from the leaves of S. trilobata, which exhibited an MIC
of 6,250 µg/mL for this bacterial species. The same
assessment was conducted by SOARES et al. (2008)
using the crude hydroalcoholic extract from the dried
bark of Stryphnodendron adstringens, with MIC values
>400 μg/mL. Another studied bacterium in the present
study was S. marcescens, which was inhibited with an
MIC of 12,500 µg/mL. In another study conducted by
NURTJAHJA et al. (2013), S. marcescens was shown
to be susceptible to the methanolic extract from the
leaves of Callicarpa candicans.
In the present study, in addition to the
cultures that were inhibited, there were also some
that exhibited resistance against the tested extract,
namely S. Typhimurium, K. pneumoniae, and P.
aeruginosa. According to BATISTA et al. (2011) and
NURTJAHJA et al. (2013), plant products exhibit a
stronger antimicrobial activity against Gram-positive
bacteria, and the antimicrobial activity of the extracts
may be attributed to the presence of secondary
metabolites, such as alkaloids, flavonoids, terpenoids,
and phenolic compounds.
CONCLUSION
Results obtained in the present study
indicated that the crude hydroalcoholic extract from
the leaves of S. trilobata, which contains terpenes and
flavonoids among its major secondary metabolites,
exhibit antimicrobial activity against bacterial
cultures isolated from human and dog skin.
ACKNOWLEDGMENTS
The authors wish to thank the Brazilian federal
government’s Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior(CAPES), the Fundação de Amparo a Ciência
e Tecnologia do Estado de Pernambuco.(FACEPE), and the
Universidade Federal Rural de Pernambuco(UFRPE) for their
financial support and help in the development of this research.
DECLARATION OF CONFLICT OF
INTEREST
The authors declare no conflict of interest. The
founding sponsors had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of
the manuscript, and in the decision to publish the results.
AUTHORS’ CONTRIBUTIONS
AGBL, LRME and JEN conceived and designed
experiments. AGBL, ETNF, APO, REFA, MMC and JRGSA
performed the experiments. AGBL carried out the lab analyses. AGBL
and LRME performed statistical analyses of experimental data. AGBL
and LRME prepared the draft of the manuscript. All authors critically
revised the manuscript and approved of the final version.
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